G-protein coupled receptors (GPCR) are present in most if not all cells in the human body and play vital roles in cell communication and survival. They are targets for more than 50% of currently marketed pharmaceuticals. Therefore, significant emphasis has been placed on understanding molecular mechanisms governing GPCR function. GPCR are believed to form dimeric or oligomeric complexes. However, little is known about how dimer/oligomer biogenesis occurs and how it regulates GPCR function. The purpose of this proposal is to address fundamental questions regarding GPCR dimer/oligomer formation using the serotonin 5-HT2C receptor as a model system. The 5-HT2C receptor is widely distributed throughout the brain and is a target for drugs used to treat anxiety, depression, schizophrenia, and obesity. It is the goal of the proposed study to determine if native GPCRs, endogenously expressed in their natural physiological environment, function as monomers, dimers, tetramers or higher order complexes. This will be accomplished using state-of-the-art confocal microscopy combined with fluorescence correlation spectroscopy (FCS) and bimolecular fluorescence complementation that will allow direct visualization of 5-HT2C receptors and provide evidence for homodimerization of native receptors expressed in living primary cell cultures. Native 5-HT2C receptors endogenously expressed in primary choroid plexus epithelial cells and in primary hippocampal neurons will provide the model systems for FCS analysis. By measuring the fluctuations in fluorescence intensity of fluorescent molecules diffusing through a very small volume, FCS allows single molecule detection sensitivity and provides information about the diffusion properties as well as the number of fluorescent molecules traveling together within a protein complex. Fluorescent Fab fragments generated from a monoclonal antibody specific for an extracellular domain of the 5-HT2C receptor will be used to label 5-HT2C receptors in live primary cultures. FCS experiments will be performed to determine if 5-HT2C receptors, in their native environment are expressed as monomers, dimers, tetramers and/or higher order complexes. These studies will begin to elucidate the true physiological/structural characteristics of the 5-HT2C receptor. Additional clues as to the role of dimer/oligomer formation in GPCR function may be provided by FCS studies performed in the absence and presence of serotonin. These studies will be among the first to provide information about the dimeric/oligomeric nature of endogenously expressed GPCR in their intact, native cellular environment. An understanding of the overall GPCR architecture is crucial to understanding GPCR function in normal and pathological conditions and to the development of novel therapeutic agents. PUBLIC HEALTH RELEVANCE: G-protein coupled receptors (GPCR) are present in all cells in the human body, they play vital roles in cell communication and survival, and are targets for more than 50% of currently marketed pharmaceuticals. The studies in this proposal will begin to elucidate how GPCR come together to form higher order complexes (dimers/oligomers) and how this process regulates receptor function. Understanding the mechanisms involved in GPCR activation is crucial to understanding receptor function in normal and pathological conditions and to the development of novel therapeutic agents.